Methods and system for image guided cell ablation with microscopic resolution

ABSTRACT

The invention provides systems and method for the removal of diseased cells during surgery

RELATED APPLICATIONS

This Application claims the benefit under 35 U.S.C. §120 of U.S.application Ser. No. 13/314,799, entitled “METHODS AND SYSTEM FOR IMAGEGUIDED CELL ABLATION WITH MICROSCOPIC RESOLUTION” filed on Dec. 8, 2011,which is herein incorporated by reference in its entirety. ApplicationSer. 13/314,799 claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application Ser. No. 61/421,077, entitled “METHODS ANDSYSTEM FOR IMAGE GUIDED CELL ABLATION WITH MICROSCOPIC RESOLUTION” filedon Dec. 8, 2010, which is herein incorporated by reference in itsentirety.

FIELD OF INVENTION

The present invention relates generally to the ablation of abnormalcells guided by imaging of such cells during surgical procedures.

BACKGROUND OF INVENTION

A major challenge of oncology surgery is removing cancer cells from thetumor bed with certainty. Residual cancer, which refers to cancer cellsleft behind after the initial resection surgery, can lead to localrecurrence, increased rates of metastasis, and poorer outcomes.Currently, there is a high rate of secondary surgeries because cancercells are found at the margins of the resected mass duringpost-operative pathological analysis of the tumor. For example, 50% ofbreast conserving lumpectomies (Mullenix et al., Am. J. Surg.,187:643-646, 2004), 35% of limb-sparing sarcoma surgeries (Zomig et al.,Br. J. Surg., 82:278-279, 1995), and 37% or radical prostatectomies(Vaidya et al., Urology, 57:949-954, 2001) fail to completely removecancer cells during the initial surgery. One of the leading causes ofnot being able to remove all the cancer cells in the tumor bed is thelack of an intraoperative visualization technology that can guide thesurgeon to identify and remove the diseased cell. In many cases,effective and total resection of cancers in organs is furthercomplicated because essential adjacent structures need to be spared (forexample brain surgeries or other surgeries where important nerves orblood vessels are nearby).

Standard assessment of a resection is performed by inking the outside ofthe excised tissue, freezing it and then examining the edge of specimensections by light microscopy (known as frozen section analysis). Thepresence of tumor cells at the inked margin, which is referred to as apositive margin, indicates that tumor cells remain behind in the tumorbed. Although margin assessment of a frozen section can take placeduring surgery, time constraints normally limit this assessment to smallareas of the tumor. Therefore, this approach is prone to sampling error.The remaining excised tissue is fixed in formalin and it may takeseveral days before the pathologist can complete the analysis toidentify a positive margin. If a positive margin is identified, patientsmost often require a repeat surgical resection, leading to increasedpatient morbidity and higher healthcare costs. Other intraoperativecancer detection technologies have been developed includingradio-frequency (RF) spectroscopy analysis of the surface of resectedtumors (Allweis et al., Am. J. Surg., 187:643-646, 2004), Raman andelastic scattering spectroscopy (Bigio et al., J. Biomed. Opt.5:221-228, 2000) and tissue autofluorescence (Demos et al., J. Biomed.Opt., 9:587-592, 2004). However, each of these technologies lacks theresolution, sensitivity and ease of use required for rapid assessment ofmicroscopic residual cancer within the entire tumor and does not providemeans of tissue removal.

A common method used to destroy cells in situ is laser ablation therapy.Laser ablation therapy refers to the destruction of tissue by deliveringheat in the form of light into a small volume. Typically, the laserlight is presented in short pulses to reduce damage and overheating ofsurrounding healthy tissue. The amount of tissue being ablated iscontrolled by the size of the laser focal spot (0.2-3 mm in diameter),intensity and duration of exposure. At the focal spot, temperatures willreach 100.degree. C. which causes vaporization of the tissue due toevaporation of interstitial water (Gough-Palmer et al., Laryngoscope,116:1288-1290, 2006). At about 1.5 diameters, temperatures reaching50.degree. C.-54.degree. C. will induce instant cell death, rapidcoagulative necrosis, and immediately cauterize the wound limiting theblood loss to a minimum (Goldberg et al., Acad. Radiol., 3:212-218,1996).

To reach the desired depth of ablation, the wavelength of the laserlight has to be carefully chosen. For example, a potassium titanylphosphate laser (KTP) producing light at a 532 nm wavelength istypically used for ablation of tissue limited to surface treatment (forexample, skin cancer and tumors at the periphery of organs), as itsdepth of penetration is only 900 .mu.m. Carbon dioxide lasers are alsoused for surface ablation as its 10.6 .mu.m wavelength is heavilyabsorbed by water inside tissue limiting its penetration depth toapproximately 300 .mu.m. For ablation of diseased tissue below thesurface, Nd:YAG lasers, operating at a wavelength of 1064 nm, providepenetration depths up to 15 mm (Reinisch, Otoralyngol. Clin. North. Am.,29:893-914, 1996).

Laser ablation procedures are usually non- or minimally-invasive andguided by standard imaging techniques. Currently, laser ablation hasbeen used intraoperatively to remove visible cancer nodes in lungtumors, unresectable liver metastasis, small breast cancers andlaryngeal cancers. However, ablation therapy lacks cellular resolutionbecause it is often limited by the spatial resolution provided by theguiding imaging techniques; thus, it can easily leave millions of cancercells behind. For example, the Gamma Knife unit used in brain surgeryhas a theoretical accuracy of 0.2 mm but it is limited by the imagingresolution of 2 mm and positioning and excision accuracy of the surgeon.

Thus, a need exists for an intraoperative and real-time cancer celldetection and therapy device at a single-cell level to ensure thoroughexamination of the tumor bed for residual cancer while providingguidance for additional tissue removal. A single-cell image detectiontechnology could be use to guide an automatic cell ablation system todestroy the cancer cells as soon as they are detected. The combinedsystem will give the surgeon the ability to remove cancer cells at anunprecedented single cell level while providing a minimum impact on thehealthy tissue. This will address the difficulty of removing residualcancer in complicated open and endoscopic surgeries such as brain,sarcoma, and colon.

SUMMARY OF INVENTION

The invention is based on a system which is capable of detectingabnormal cells at a single cell resolution and treating the abnormalcells with laser ablative therapy. The laser and imaging system arepreferentially detecting and treating surface cells. Although abnormalcells that are cancerous in nature are ideally targeted, the system andmethods can be adapted to other abnormal cells or tumor-associated cellsas well. Alternatively, other energy sources can be used in place of thelaser, for example radiofrequency ablation or cryoablation.

Furthermore, the invention also includes methods for intraoperativein-vivo imaging and treatment using said device. Subjects can be eitherhuman or animal. Preferably, the subject is given a fluorescent,activatable probe, administered orally, systemically, via bolusinjection, via surface application or other established method.Alternatively an antibody probe can be used or the endogenousfluorescent difference between cancer cells and healthy cells may beused without an imaging agent or probe. The probe then is allowed toreach the target tissue and is activated by the tissue at the targetlocation. During the surgical operation, the diseased tissue is exposedby the surgeon and the bulk diseased tissue is removed, if possible. Theimaging device is then used to identify and treat the residual abnormalcells.

In one aspect the invention provides an in vivo method of treatingabnormal cells by administering a composition comprising a molecularimaging probe to a tissue of a subject and obtaining an in situ image ofthe tissue where the image allows for the detection of one or morediseased cells, if present in the tissue and treating the diseased cell.The subject is a mammal such as a human.

The composition is administered systemically to the subject or appliedto the surface of the tissue, such as by spraying or painting.Alternatively the composition is administered on a film or sponge.

In some embodiments the cells are treated with light energy, such as alaser. Cells are ablated by a laser by locating one or more diseasedcells in situ using an imaging system, transferring the location of thediseased cell to a laser guiding system to move the laser over thetarget cell for ablation, imaging the actual location of the laser usingthe imaging system to provide spatial feedback to the laser guidingsystem, adjusting the location of the laser if necessary based on theaforementioned feedback and ablating detected diseased cell(s).Optionally, the method includes an additional feedback algorithm afterthe ablation of diseased cells to post-image the treatment tissue andverify that the diseased cells have been correctly treated. The imagingsystem is a single-cell resolution imaging system. The laser guidingsystem consists of one or more galvanometer minors, MEMS minors,acousto-optic deflectors, micromirrors, acousto-optic modulators used asdeflectors, piezo-electrial mirrors, electro-optical deflectors,polygonal mirrors, or planar minors on a rotating shaft.

The molecular probe can be any molecule that gives us a contrast betweenthe diseased cells and normal tissue and can include either activated,ligand or clearance differential. The activated can be activated byenzymes and can be a flourochrome plus a quencher or two flourochromesin a self-quenching configuration. A ligand based probe would be forinstance a flourochrome together with a targeting antibody. A clearancedifferential probe would be a molecule with a fluorescent label and apharmacokinetic modifier that clears the probe preferentially from thehealthy tissue leaving the cancer cells and/or tumor associatedinflammation cells labeled.

The molecular imaging probe is activated by enzymes. In another aspectthe imaging probe contain one or more fluorochromes and one or more darkquenchers. Exemplary fluorochromes include Cy3, Cy3.5, Cy5, Alexa 568,Alexa 546, Alexa 610, Alexa 647, ROX, TAMRA, Bodipy 576, Bodipy 581,Bodipy TR, Bodipy 630, VivoTag 645, and Texas Red.

Exemplary dark quenchers include a QSY quencher, a dabcyl quencher, anIowa Black quencher, and a Black Hole quencher. The QSY quencher isQSY21, QSY7, QSY9, or QSY35. The Iowa Black quencher is Iowa Black FQ orIowa black RQ. In some aspects the imaging probe is in the visible lightspectrum of 350-670 nm. Optionally, imaging probe includes apharmacokinetic modifier. The probe is optimally imaged at less than 2hours after administration. Alternatively, the probe is optimally imagedat between 12 and 36 hours after administration.

In some aspects the molecular imaging probe contains a targeting moietyand an imaging moiety. A targeting moiety binds specifically to CD20,CD33, carcinoembryonic antigen (CEA), alpha fetoprotein (AFP), CA125,CA19-9, prostate specific antigen (PSA), human chorionic gonadotropin(HCG), acid phosphatase, neuron specific enolase, galacatosyltransferase II, immunoglobulin, CD326, her2NEU, EGFR, PSMA, TTF1, Muc,immature glycoslytaion, an EMT marker, a cathepsin, or an enzyme. Theimaging moiety is a fluorochrome such as Cy3, Cy3.5, Cy5, Alexa 568,Alexa 546, Alexa 610, Alexa 647, ROX, TAMRA, Bodipy 576, Bodipy 581,Bodipy TR, Bodipy 630, VivoTag 645, and Texas Red.

The diseased cell is within 1 cm from the surface. The diseased cell isfor example a cancer cell, a central nervous cell, a cardiac cell, abone cell, a tendon cell, or a muscle cell.

Also included in the invention is a medical imaging and treatment systemcontaining:

-   -   (a) an excitation source configured to cause an object having a        plurality of cells to emit and fluoresce light;    -   (b) an optical receptor configured to receive the light from the        object;    -   (c) an image processor;    -   (d) an energy source sufficient for destroying one or more        cells; and    -   (e) a feedback system configured to detect the condition of each        cell and apply treatment to detected diseased cells.

The image processor contains a field of view (FOV) substantially greaterthan a diameter of a cell of the object and an analysis resolutionsubstantially matched to the diameter of a cell of the object andconfigured to receive and analyze the light corresponding to each cellin the FOV.

The cells are treated using light energy. In some aspects the lightenergy is delivered by a plurality of lights. For example, an opticalfiber bundle collects and distributes light to the cells.

In another embodiment the cells are treated using laser ablation, radiofrequency ablation or cryo-ablation. The laser is controlled by a laserguiding system comprising of one or more galvanometer mirrors, MEMSmirrors, acousto-optic deflectors, micromirrors, acousto-opticalmodulators used as deflectors, piezo-electrial mirrors, electro-opticaldeflectors, polygonal mirrors, or planar mirrors on a rotating shaft.

A single light source may be used for both the fluorescent excitationand then a higher power setting would provide the ablation function.

The light energy is between 100 nm and 2500 nm. In some aspects thelight energy is pulsed. For example, the pulse is for duration less than100 ns. In some aspects, the light energy imparts delayed cell death.

The feedback system consists of:

-   -   (a) locating the diseased cell in situ using an imaging system;    -   (b) transferring the location of the diseased cell to a laser        guiding system to move the laser over the target cell for        ablation    -   (c) imaging the actual location of the laser using the imaging        system to provide spatial feedback to the laser guiding system    -   (d) adjusting the location of the laser if necessary based on        the aforementioned feedback    -   (e) ablating detected diseased cell(s).

Optionally the system further contains an additional feedback algorithmafter the ablation of diseased cells to post-image the treatment tissueand verify that the diseased cells have been correctly treated. In someembodiments the system contains a fluid reservoir which flushes cellarea with fluid to remove debris created during ablative process.Additionally, in some embodiments the system contains a fluid reservoirwhich flushes cell area with fluid to cool tissue as ablative heatdestroys abnormal cells, preventing excess treatment to healthy tissue.

In another embodiment, the method does not require an imaging probe.Instead, an imaging method (e.g. fluorescence, spectroscopy, or otherimaging technique) is used to determine which cells to ablate.

In another embodiment, the system requires a light source to image thetissue. In some aspects, the system does not excite an imaging agent.

As used herein, “probe” means an identifiable molecule which is used todetect the presences of other molecules.

As used herein, “fluorochrome” means a molecule which becomesfluorescent by absorbing energy (light) at one or more specificwavelengths by exciting ground-state electrons into a higher energylevel and then emitting energy (light) at one or more slightly differentwavelengths when the excited electrons return to the ground-state energylevel.

As used herein, “dark quencher” means a molecule which absorbs lightradiation at one or more specific wavelengths and dissipates the energyabsorbed in the form of heat; thus, a dark quencher does not emitfluorescent light.

As used herein, “pharmacokinetic modifier” means a molecule which isattached to the molecular imaging probe which inhibits undesiredbiodegradation, clearance, or immunogenicity of the probe.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice of the present invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are expressly incorporated byreference in their entirety. In cases of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples described herein are illustrative onlyand are not intended to be limiting.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: Optical layout of laser and imaging system. Fluorescenceexcitation is provided by a ring of LED. Lenses L1 and L2 collect theimage of the specimen plane and relay it to lens L3, which focuses itonto the CCD camera. Band-pass filter F1 blocks all wavelengths exceptthe fluorescence emission of the target. A laser diode provides highintensity illumination to induce photobleaching of the target tosimulate ablation. The laser light is actively directed by a2-dimensional set of galvo-mirrors. Lens L4 adjusts the focus of thelaser beam at the specimen plane. The laser light is brought into theoptical axis by reflecting off a 50-50 beam splitter (M1). Duringexposure of high laser intensity, shutter Si prevents light fromdamaging and/or saturating the CCD.

FIG. 2: Light source array used to ablate tissue. Each individual lightsource is in close enough proximity to the tissue to kill one or morecells.

DETAILED DESCRIPTION OF INVENTION

The present invention relates to the design of a system which is able tosimultaneously detect abnormal cells and treat the cells with a laserablation. The invention applies to the removal of diseased cells insurgery. Cancerous tissue removal is one such application though not theonly one as abnormal cells in various central nervous system disorders(e.g. Parkinson's disease) or various cardiovascular system disorders(e.g. ischemia), or various orthopedic disorders (e.g. osteoporosis)could be a target for removal.

FIG. 1 shows a schematic of one example of such a system. The imagingsystem consists of a number of components including an excitation sourcesuch that one or more diseased cells are caused to emit fluorescentlight, an optical receptor to receive the emitted light from thediseased cells, an image processor, an energy source for destroyingdetected diseased cells, and a feedback system for detecting thecondition of each cell and applying treatment to the detected diseasedcells.

In the image detection portion of the system of FIG. 1, fluorescenceexcitation is provided by a ring of LED. The target abnormal specimencontains an activated fluorescent probe such that the probe is excitedby one wavelength of light and emits a second, distinct wavelength.Lenses L1 and L2 collect the image of the specimen plane and relay it tolens L3, which focuses it onto the charge-coupled device (CCD) camera(or alternatively an avalanche photodiode: APD array or complementarymetal-oxide semiconductor: CMOS). Band-pass filter Fl blocks allwavelengths including the excitation fluorescence of the LED ring andallows the fluorescence emission of the target to reach the camera.

The image detection portion of the system provides a wide field of view(FOV) with an analysis resolution substantially matched to the size of acell by matching a given cell with one or more pixels of a CCD, APD, orCMOS array such that the FOV of any pixel is one cell or less. Thisprovides a desirable photon flux rate (photons/sec-area) and desirablycontrols the background emission (auto fluorescence) which, along withthe dark count, determines the signal-to-noise ratio of the instrumentand its sensitivity. If the field of view of a pixel contains severalcells and only one is a cancer cell that has illuminated molecularprobes, the average photon flux rate to the pixel will be reduced andthe ratio of the signal-to-background noise will be, likewise, reduced.Furthermore, if multiple cancers cells are closely spaced, the devicewill still be able to differentiate individual cells.

For the laser ablation portion of the system of FIG. 1, the laser lightis actively directed by a 2-dimensional set of galvo-mirrors. Lens L4adjusts the focus of the laser beam at the specimen plane. The laserlight is brought into the optical axis by reflecting off a 50-50 beamsplitter (M1). During exposure of high laser intensity, shutter 51prevents light from damaging and/or saturating the CCD.

The pair of galvo-mirrors will provide 2-dimensional control of thelocation of the laser in the specimen plane. Galvanometer-mirrors (orgalvo-mirrors) consist of a galvanometer-based scanning motor with aminor mounted on the motor shaft and a detector that provides feedbackon the current angular position to the controller. Galvo-minors have aprecision in the range of 15 .mu.radians, a step response time of 300.mu.s and a small angle bandwidth of 1 kHz, thus providing fast controlof the laser. These minors are usually employed commercially for fastlaser engraving and bar code reading. Galvo-mirrors are also used inresearch for laser scanning applications such as optical tweezers andsome types of confocal microscopy set ups.

The path length between the galvo-mirrors and the laser focusing lens(L4) can be adjusted in order to achieve full coverage of the camerafield of view at the largest angular deflection (laser location isproportional to the path length multiplied by tangent of the angulardeflection of galvo-mirrors). The laser will be brought into the opticalimaging path via a 50-50 beam splitter to reflect 50% of the light onthe specimen plane. In contrast to a dichroic mirror, this 50-50 beamsplitter also allows some of the laser light in the specimen plane toreach the camera, which can be used for the imaging and a feedbackalgorithm. The distance between lenses L4 and L2 can be set such that a100 .mu.m laser spot diameter (or any other desired spot diameter) canbe obtained at the specimen plane—this distance can be mathematicallydetermined once all the parameters involved (input beam diameter andfocal lengths of all the lenses) are determined.

A calibration curve to correlate the beam angular deflection by thegalvo-mirrors with the x-y location of the laser beam in pixelcoordinates can be empirically determined. The galvo-mirrors can bescanned over a range of angles while an image is acquired at eachangular step to determine the pixel location of the beam spot. To avoiddamage to the camera chip and signal saturation, the laser intensity canbe set at a reduced power during this calibration. Also, thefluorescence emission filter (F1) can be removed to avoid filtering outthe laser light before reaching the camera. The calibration curve can befitted to a suitable polynomial function in the form of (.alpha.,.beta.)=f(xp, yp), where .alpha. and .beta. correspond to the angulardeflection of each galvo-mirror, respectively, and xp and yp correspondthe x- and y-coordinates in camera pixels.

Once the laser calibration curve is known, a detected abnormal cell canbe treated. The camera pixel locations of cell are input into thecalibration equation (.alpha., .beta.)=f(xp, yp) to obtain thegalvo-mirror angular deflection required to direct the laser (in lowpower mode) into the correct specimen plane location. At this time, thelaser spot can be imaged to determine its actual location in thespecimen plane using the detection system. The actual location of thelaser relative to the targeted pixel can be used to provide feedback tothe guiding algorithm to adjust the laser location, as necessary.

Because of experimental errors and thermal drift, the calibrationbetween pixel location and the laser angular deflection might experiencevariations that limit the spatial accuracy of the system. Thus, aclosed-loop control system that feedbacks the actual location of thelaser spot relative to the targeted pixel can be implemented. Once thelaser is positioned over the right location, an automatic control canswitch the laser to high power mode for a predetermined time duration,inducing the cells to pre-maturely die. During the high power exposureperiod, the camera chip will be protected by closing a shutter, S1,placed in front of the camera.

To reach the desired depth of ablation, the wavelength of the laserlight has to be carefully chosen. For example, a potassium titanylphosphate laser (KTP) producing light at a 532 nm wavelength istypically used for ablation of tissue limited to surface treatment (forexample, skin cancer and tumors at the periphery of organs), as itsdepth of penetration is only 900 .mu.m. Carbon dioxide lasers are alsoused for surface ablation as its 10.6 .mu.m wavelength is heavilyabsorbed by water inside tissue limiting its penetration depth toapproximately 300 .mu.m. Alternatively, an ArF excimer laser at around a248 nm wavelength can be used to treat cells at around 30 .mu.m depthand an XeCl excimer laser at around 308 nm wavelength can be used totreat cells around 50 .mu.m depth (Vogel et al., Chem Revv,103:577-644). A 2.01 .mu.m Cr:Tm:YAG laser has a depth of around 170.mu.m and a 2.12 .mu.m Cr:Tm:Ho:YAG laser has a depth of around 350.mu.m (Vogel et al., Chem Revv, 103:577-644). For ablation of diseasedtissue below the surface, Nd:YAG lasers, operating at a wavelength of1064 nm, provide penetration depths up to 15 mm (Reinisch, Otoralyngol.Clin. North. Am., 29:893-914, 1996). Lasers in the 1-5W power range aresufficient for ablating tissue with a single nanosecond pulse (Reinisch,Otoralyngol. Clin. North. Am., 29:893-914, 1996). Other powers can beused with accordingly shorter and longer duration treatment periods.

Importantly, quick treatment periods are preferred to ensure thatdetection and treatment is co-localized and device/patient movement doesnot affect the treatment accuracy. Several factors affect the speed ofthe system: (1) image acquisition time (exposure), (2) image processing,(3) ablation time, (4) laser positioning, and (5) number of ablationtargets. With 25 million spots (5 cm.times.5 cm of cells) the cumulativelaser duration will be about 0.2 seconds if the pulse duration is 1 ns.The speed of the system is mostly limited by a combination of how fastthe laser can be moved between targets and how many targets have to beablated.

Using galvanometer driven mirrors, a laser beam can be scanned over a500.times.500 pixel in 33 ms (Pawley, Handbook of Biological ConfocalMicroscopy, 3rd ed) or about 3.3 seconds to ablate 25 million pixels. Weexpect that in 3.3 seconds, loss of registration would become a problem.To overcome this limitation, we have developed an algorithm to dividethe field of view into several small regions to be independently ablatedwithin 0.5 s. Fiducial marks can be generated by the laser to identifyeach region so the system can automatically detect which regions havebeen already ablated before moving to the next one. Also, the fiducialmarks will allow correct repositioning of the device in case it is movedbefore ablation has been completed.

Preferably, the pulse duration of the laser and the mirror movement isquick enough to enable image detection and ablation to be co-registered.

A low power laser can also be employed in this invention such thatdelayed treatment is provided. Specific ablation energy can be deliveredto impart nucleic damage to the cell, inducing apoptosis at a futuretime. DNA absorption is centered around a peak of 260 nm (Vogel et al.,Chem Revv, 103:577-644). Alternatively, lasers at other UV and IRwavelengths have been shown to impart DNA damage by various mechanisms(Kong et al., Nucleic Acids Res., 37:e68, Rogakou et al., J. Cell Biol.,146:905-915). Lasers at low enough powers to not induce instantaneousvaporization may be used to impart delayed treatment effects.

Ideally, a laser with appropriate qualities to treat cells at or nearthe surface of the tissue while sparing deep tissue treatment ispreferred in this application. Furthermore, lasers with limitedtreatment zones are preferred so as not to affect nearby healthy tissue.

To ensure that cell ablation has occurred, a second feedback algorithmcan be implemented to detect when the fluorescence of the current targethas been extinguished should the treatment energy be sufficient to killnot only the cells, but also the imaging probe as well. After theinitial exposure to high power laser is completed, the laser can beswitched off, the shutter in front of the camera can be opened and theemission filter can be placed in front of the camera. A second image ofthe specimen plane can be acquired and compared in a pixel-by-pixelbasis with the image acquired pre-ablation. If the current target isstill emitting fluorescence, the system can be switched to high powermode for a second exposure. The process can be repeated as necessary.When the current target no longer emits, the nearest fluorescent targetcan be selected as a new target based on the latest image acquired, andthe procedure can be repeated.

With this feedback algorithm, it is important to note that anyunactivated imaging agent in healthy tissue may actually be activatedwith the laser in a “heat affected” zone. A “heat affected” zone wouldbe the tissue which is not completely denatured, but was subjected toabove normal temperatures and is located in between that tissuecompletely ablated and that tissue which was not affected by the laser.Consequently, the laser should be chosen to apply small treatment zoneswith minimal “heat affected” zones.

Because the tissue exposed during the surgery could have an irregularsurface which is not flat, or cancer cells may be one or two layers ofcells below the surface, the system can adjust the location of the laserfocus to control the depth of ablation. Because the depth of focus ofthe laser depends on the imaging system properties (numerical aperture,focal length of lenses, diameter of lenses, path length), the opticaldesign can be manipulated to provide an ablation focal depth ofapproximately 500 .mu.m.

It is possible that the burned tissue or debris generated by laserablation may interfere with the signal of nearby cells, limiting theirdetection. The proposed invention also includes an embodiment with acontinuous fluid wash which can be applied to the surface to remove anyinterfering debris. The proposed fluid wash can alternatively be used tocool the tissue such that the focal ablation heats and affects only thedesired location while providing minimal impact on healthy tissue.

In other embodiments of the invention, the laser beam is directed to thetreatment site by controlling the set of mirrors through the use of astepper motor or using MEMS mirrors instead of the galvo-mirrors. In afurther embodiment, the laser beam can be moved by controllingacousto-optic deflectors. The invention also includes a method ofdirecting the laser beam treatment using micro-mirrors. Anacousto-optical modulator used as a deflector, a piezo-electric mirror,an electro-optical deflector, a polygonal mirror, or a planar mirror ona rotating shaft can also be used to direct the laser beam. Any methodor combination of methods used to modify the mirror angles such that thelaser light energy is directed onto the desired treatment tissue issuitable for this application.

In other embodiments of this invention, the light source is a singlefixed bulb instead of an LED ring. In other embodiments of thisinvention, the tissue is ablated by using an array of light elements asshown in FIG. 2. Note that the light elements could be light emittingdiodes or individual fiber optic bundles or similar sources of energy.In this case each element is preferentially activated in order toselectively ablate the focal tissue closest to the element.

In other embodiments of this invention, the laser is replaced by aradio-frequency (RF) ablation device to similarly impart cellular death.Other embodiments include a cryo-ablation treatment device Importantly,the micro probes used to deliver the RF energy or cryoablation treatmentshould be small enough such that a minimal treatment zone is resultant.An array of such micro RF or cryo probes could be controlled easily ifapplied to the surface of the tissue in place of the light source arraypictured in FIG. 2. Each micro probe could be individually controlledcreating a targeted treatment area dependent upon the image detectionsystem.

The present invention also includes methods for imaging and treatingabnormal cells during an intraoperative surgical procedure. In oneaspect, the invention provides a method for spatially determining tissueheterogeneity in a subject undergoing surgery or a minimally invasiveintervention by administering a molecular imaging probe to the subjectand obtaining an in situ image of the tissue. The image allows for thedetection of a diseased cell, if present in the tissue and treatment ofdetected diseased cells. Furthermore, the method could entail repeatingthe imaging and treatment steps until no diseased cell is detected inthe tissue.

The molecular probe is any molecule that gives us a contrast between thediseased cells and normal tissue and can include either activated,ligand or clearance differential. The activated can be activated byenzymes and can be a flourochrome plus a quencher or two flourochromesin a self-quenching configuration. A ligand based probe would be forinstance a flourochrome together with a targeting antibody. A clearancedifferential probe would be a molecule with a fluorescent label and apharmacokinetic modifier that clears the probe preferentially from thehealthy tissue leaving the cancer cells and/or tumor associatedinflammation cells labeled.

In one embodiment, the molecular imaging probe could be administeredsystemically to the subject or alternatively to the surface of thetissue. Surface administration includes for example spraying orpainting. Optionally the molecular imaging probe is administered on afilm or sponge. The probe can also be administered orally, parenterally,via bolus injection, or other method used commonly in the surgicalpractice. Other forms of acceptable administration includesubcutaneously, intracutaneously, intravenously, intramuscularly,intraarticularly, intraarterially, intrasynovially, intrasternally,intrathecally, intralesionally, by intracranial injection or infusiontechniques, by inhalation spray, rectally, nasally, buccally, vaginally,via an implanted reservoir, by injection, subdermally,intraperitoneally, transmucosally, or in an ophthalmic preparation.

In one embodiment, the molecular imaging probe is an activatable probe.An activatable imaging probe exhibits no fluorescence emission or itsfluorescence emission is quenched in its nominal configuration, but itsfluorescence emission is typically released upon enzymatic cleavage ofits backbone. Activatable imaging probes have been specifically designedto target enzyme families with well established catalytic mechanismsincluding proteases, kinases, lipases, glycosylases, histone deacylases,and phosphatases. Optimally, the activatable imaging probe targets anenzyme that is either preferentially expressed in cancer cells or isupregulated in cancer cells. Thus, the imaging moiety is only active incancer cells, allowing for discrimination between cancer and normaltissue. For example, the probe targets an enzyme in the cysteineprotease family (e.g., caspase), cysteine cathepsin family (e.g.cathepsin B), serine protease family or the aspartic protease family.

In one embodiment of this invention, the imaging moiety is active incancer cells as well as tumor-associated cells (eg. tumor-associatedmacrophages) allowing for discrimination between these cells and normaltissue.

In one embodiment of this invention, the probe is constructed of one ormore fluorochromes quenched by each other or quenched through the use ofdark quencher molecules, attached together with an enzyme activationsite and a pharmacokinetic modifier Importantly, the pharmacokineticmodifier is adjusted to optimize the administration-to-imaging timespread. After cleavage of the enzyme activation site, the fluorochromesand quenchers are spatially separated allowing fluorescence excitationand detection of the fluorochromes.

A dark quencher emits no fluorescence, but absorbs fluorescence fromnearby fluorochromes. Suitable dark quenchers include but are notlimited to: QSY (diarylrhodamine derivatives) type quenchers (e.g.QSY21, QSY7, QSY9, QSY35), dabcyl type quenchers, Iowa black FQ and RQquenchers, and Black Hole quenchers.

The probe fluorochrome is chosen from a group of available compounds inthe 350-670 nm visible spectrum to preferentially image cells at or nearthe tissue surface (within 1 cm) while ignoring deep tissue emission.Suitable examples of fluorochromes in the visible light spectrum whichcould be used include but are not limited to: Cy3, Cy3.5, Cy5, Alexa568, Alexa 546, Alexa 610, Alexa 647, ROX, TAMRA, Bodipy 576, Bodipy581, Bodipy TR, Bodipy 630, VivoTag 645, and Texas Red.

Imaging moieties in the visible light spectrum of 350-670 nm arepreferred to selectively view cells at or near the surface (within 1 cmfrom the surface) and exclude deep tissue emission since the imageddepth of tissue penetration increases as the imaging wavelengthincreases. Near infrared wavelengths are not needed for this probeapplication since deep tissue penetration of the energy is not desired.Tissue absorbance and autofluorescence is high between 400 nm and 500 nmwhile slowly dropping off around 570 nm Thus the longer wavelengths inthe visible spectrum are preferred for this application.

In some embodiments, the molecular imaging probe includespharmacokinetic modifiers of adjustable molecular weight and size whichallows the bio-distribution and diffusion rate of the molecular imagingagent to be controlled. For example, polyethylene glycol (PEG) and/ordextran can be used as a pharmacokinetic modifier because its chainlength, and thus molecular weight, can be precisely controlled andreadily conjugated to the imaging probe. Other forms of PEG that can beused are polyethylene oxide (PEO) or polyoxyethylene (POE). Othersuitable pharmacokinetic modifiers are methoxypolyethylene glycol(MPEG), methoxypolypropylene glycol, polyethylene glycol-diacid,polyethylene glycol monoamine, MPEG monoamine, MPEG hydrazide, MPEGimidazole, copolymers of polyethylene glycol and monoxypolypropyleneglycol, branched polypropylene glycol, polypropylene glycol, andpolylacic-polyglycolic acid. Optionally, any fatty acid, lipid,phospholipid, carbohydrate, sulfonate, polysulfonate, amino acid, orpeptide can be used as a pharmacokinetic modifier to tune thebiodistribution of the molecular imaging probe.

Importantly, the size and weight of pharmacokinetic modifiers can beadjusted to modify the kinetics of the probe. Smaller sizes and weightsare more useful for surface applications where the probe is directlyapplied to target tissue and immediate imaging is necessary. Largersizes and weights allow for the probe to be injected and travel to thetarget tissue via vascular routes. Further small modifications of thesize and weight can be used to adjust the retention time of the probe inthe target tissue. In some embodiments the molecular probe needs toreside in the target tissue at least 1 hour and up to 48 hours.Optionally the pharmacokinetic modifiers are optimized to allow imagingwithin 2 hours of administration or between 12 and 36 hours post probeadministration. In some embodiments of the present invention, probeswith PEG attachments around 2,000 g/mol and between 20,000 g/mol and40,000 g/mol are preferably used to target surface applications (smallmolecular weight PEG) and injectable (larger molecular weight PEG)versions of the molecular imaging probe. Although these are examples,other PEG sizes and different pharmacokinetic modifiers can be used. PEGmolecules are typically available in a large array of molecular weightsfrom 300 g/mol to 10,000,000 g/mol.

Preferably, the pharmacokinetic modifier is between 500 g/mol and100,000 g/mol. In another embodiment of the present invention, theimaging probe consists of a targeting moiety and an imaging moiety. Theimaging moiety can be a fluorochrome and suitable examples offluorochromes in the visible light spectrum which could be used includebut are not limited to: Cy3, Cy3.5, Cy5, Alexa 568, Alexa 546, Alexa610, Alexa 647, ROX, TAMRA, Bodipy 576, Bodipy 581, Bodipy TR, Bodipy630, VivoTag 645, and Texas Red.

The targeting moiety preferentially targets diseased tissue and affectsthe pharmacokinetics in such a way to allow for discrimination betweendiseased and healthy tissue. In some aspects the targeting moiety bindsspecifically to CD20, CD33, carcinoembryonic antigen (CEA), alphafetoprotein (AFP), CA125, CA19-9, prostate specific antigen (PSA), humanchorionic gonadotropin (HCG), acid phosphatase, neuron specific enolase,galacatosyl transferase II, immunoglobulin, CD326, her2NEU, EGFR, PSMA,TTF1, Muc, immature glycoslytaion, an EMT marker, a cathepsin, or anenzyme. The targeting moiety can specifically be designed to targetenzyme families with well established catalytic mechanisms includingproteases, kinases, lipases, glycosylases, histone deacylases, andphosphatases. Once the imaging probe binds to such a target, the probecan be cleared more quickly or less quickly than in normal tissue,allowing for the preferential discrimination between healthy anddiseased tissue. Optionally, a pharmacokinetic modifier can beconjugated to the imaging probe to further fine-tune the distributiontime properties of the imaging probe. Further, the targeting moiety canbe an antibody.

The diseased cell is a tumor cell, or alternatively can be any otherabnormal cell or marker of abnormal activity which is fluorescentlylabeled. For example, one could image tumor-associated macrophages thatare typically found surrounding tumors. In another example, one couldimage abnormal cells in various central nervous system disorders (e.g.Parkinson's disease) or various cardiovascular system disorders (e.g.ischemia), or various orthopedic disorders (e.g. osteoporosis). Itshould be understood that the above disease areas are not limiting andthat other abnormal cells could be detected.

Methods for targeting such tissue could involve conjugating associatedmolecules to the imaging probe. For example, in various cancer staging,myocardial infarctions, and certain neurological diseases, glucosemetabolism is upregulated. Imaging probes further comprising glucose ordeoxyglucose molecules could be used to target tissue in need of excessglucose. Furthermore, in malignant tissue, DNA synthesis is upregulatedand nucleotide based metabolites such as thymidine are more readilyused. Thus, an imaging probe conjugated to thymidine molecules oranalogs thereof could be useful in distinguishing cancerous tissue frombenign tissue.

In various central nervous system disorders including Parkinson'sdisease, Tourette's Syndrome, Lesch-Nyhan Syndrom, Rhett's Syndrome, andin substance abuse cases, dopamine metabolism and dopamine transportersare found in increased or decreased prevalence. Imaging these types ofdiseased cells could involve conjugating the imaging probe to L-dopa,tropanes, dopamine, and/or raclopride molecules which are moleculesinvolved with the dopamine transport into the cytosol of the cellImportantly, the imaging probe would need to pass the blood brainbarrier in this case for a systemic type probe administration.

For the cardiovascular system, the synthesis and breakdown of long chainfatty-acids is indicative of an imbalance of myocardial metabolismcommonly found in coronary artery disease, myocardial infarction,cardiomyopathies, or ischemia (Railton et al., 1987 Euro. J NucL. Med.13:63-67; and Van Eenige et al., 1990 Eur. Hearth 11:258-268). Thus toimage these diseased cells, the imaging probe could further beconjugated to one or more long chain fatty acids.

Imbalances in osteoblast activity are involved in osteoporosis,osteoblastic cancer metastases, early calcification in atheroscleroticand cancer lesions, arthritis, and otosclerosis. Since phosphonates andtheir analogs are found in higher concentrations where osteoblastactivity is increased (Zaheer et al., 2001, Nature Biotech19:1148-1154), then an imaging probe conjugated to phosphonate,phosphonate analogs, methylene diphosphonate, pyrophosphate, and/oralendronate molecules could be useful for imaging such disease states.

Typically tumors and infracted regions are hypoxic. Molecules such asnitroimidizoles and misonidazole accumulate in hypoxic areas and couldalso be conjugated to an imaging probe to preferentially discriminatehypoxic regions from normal tissue.

Alternatively, the diseased cells are detected without an imaging probe.Instead, the tissue is imaged and then treated. In this case, imagingmodalities such as spectroscopy, raman spectroscopy, optical coherenttomography, or auto-fluorescence could be used to detect those cells forfurther treatment. Furthermore, the imaging system would not need toexcite a probe.

Definitions

As used herein, “probe” means an identifiable molecule which is used todetect the presences of other molecules.

As used herein, “fluorochrome” means a molecule which becomesfluorescent by absorbing energy (light) at one or more specificwavelengths by exciting ground-state electrons into a higher energylevel and then emitting energy (light) at one or more slightly differentwavelengths when the excited electrons return to the ground-state energylevel.

As used herein, “dark quencher” means a molecule which absorbs lightradiation at one or more specific wavelengths and dissipates the energyabsorbed in the form of heat; thus, a dark quencher does not emitfluorescent light.

As used herein, “pharmacokinetic modifier” means a molecule which isattached to the molecular imaging probe which inhibits undesiredbiodegradation, clearance, or immunogenicity of the probe.

EXAMPLES Example 1

Interoperative Detection and Ablation of Cancer Tissue Currently around50% of breast cancer patients and 35% of sarcoma patients require secondtumor de-bulking surgeries because a final pathology report returns daysafter the initial surgery indicating that residual cancerous cells havebeen left within the patient. Furthermore, 25% of the final pathologyreports do not detect residual cancer cells due to sampling errorsfundamentally inherent in the process. Thus, most patients requiresubsequent medical therapy including additional radiation orchemotherapy treatment to prevent cancer recurrence and metastasisstemming from residual cancer cells. The proposed study aims toinvestigate an intra-operative method of tumor margin assessment andtreatment to ensure negative margins are obtained during the firstsurgery and additional surgeries are not required.

The intra-operative tumor margin assessment is performed by employing afluorescence-based imaging system. One day prior to procedure, thepatient is injected with an imaging agent activated by enzymesupregulated in cancer tissue. On the day of the surgery, the bulk tumoris manually resected. Then, the tumor bed is examined for residualfluorescence using a wide-field, single cell resolution imaging andablation device. Locations with high residual fluorescence aredetermined to consist of residual cancer cells and the ablation featureis activated. Intraoperative diagnosis and treatment is compared topermanent H&E staining of the tissue by a pathologist.

Protocol:

Pre-Operative

Imaging and ablation system is set up in the operating room and is instand-by mode.

Imaging agent is IV injected into a patient scheduled for bulk tumorresection surgery.

Intra-Operative

The cancer patient undergoes a standard of care tumor resection surgery.

After the gross tumor is removed from the patient, the tumor bed isimaged with the device.

Areas detected with positive fluorescence by the imaging feature arethen confirmed by the surgeon and these confirmed areas are ablated.

The tumor bed is cleaned and prepared for a second imaging and ablationsequence.

The tumor bed is re-imaged. Areas which are still fluorescent aredisplayed to the surgeon. Additional ablation on confirmed areas isperformed if so desired by the physician.

Additional tissue samples of areas with positive fluorescence prior toablation and negative fluorescence post ablation are taken and sent forhistological analysis.

A final image of the tumor bed is recorded for future reference.

The wound is closed and the patient recovers from the operation.

Post-Operative

The bulk tumor and any additional tissue samples are assessed bypathology using standard processing techniques.

Positive margins found by pathology are compared to the residualfluorescence images saved during the procedure and additional tissuetaken post ablation.

A final determination of the presence of residual cancerous tissue inthe tumor bed is made.

Data is recorded to determine if the patient requires an additionalsurgical procedure due to the presence of residual disease.

Data is recorded to determine the local re-occurrence of disease.

Data is recorded to determine the need for adjuvant care related to thesurgical site.

What is claimed is:
 1. An in vivo method of treating a subject with atumor comprising: (a) administering a composition comprising a molecularimaging probe to the subject, wherein the imaging probe is a clearancedifferential probe including a fluorescent label with optical propertiesin the visible spectrum of 350-670 nm attached to a pharmacokineticmodifier that results in the molecular imaging probe clearingpreferentially from healthy tissue leaving tumor associated inflammationcells labeled; (b) permitting the molecular imaging probe to clearpreferentially from healthy tissue leaving tumor associated inflammationcells labeled; (c) performing initial resection surgery to remove cancercells from the subject thereby creating a resection bed; (d) treatingthe resection bed with light energy and obtaining an in situ image ofthe bed using a single-cell resolution imaging system, wherein the imageallows for the detection of the tumor-associated macrophages, if presentin the resection bed; and (e) if tumor associated cells are detected in(d), performing further resection surgery to remove labeled tumorassociated cells.
 2. The method of claim 1, wherein the composition isadministered systemically to said subject or applied to the surface ofsaid tissue.
 3. The method of claim 1 wherein the molecular imagingprobe is imaged at between 12 and 36 hours after administration.
 4. Themethod of claim 1, wherein the pharmacokinetic modifier has a molecularweight of between 20,000 g/mol and 40,000 g/mol.
 5. The method of claim1, wherein the pharmacokinetic modifier selected from the groupconsisting of polyethylene glycol (PEG), dextran, polyethylene oxide(PEO), polyoxyethylene (POE), methoxypolyethylene glycol (MPEG),methoxypolypropylene glycol, polyethylene glycol-diacid, polyethyleneglycol monoamine, MPEG monoamine, MPEG hydrazide, MPEG imidazole,copolymers of polyethylene glycol and monoxypolypropylene glycol,branched polypropylene glycol, polypropylene glycol, andpolylacic-polyglycolic acid.
 6. The method of claim 1, wherein themolecular imaging probe comprises a polyethylene glycol (PEG) having amolecular weight of between 20,000 g/mol and 40,000 g/mol.
 7. The methodof claim 1, wherein the fluorescent label is selected from the groupconsisting of Cy3, Cy3.5, Cy5, Alexa 568, Alexa 546, Alexa 610, Alexa647, ROX, TAMRA, Bodipy 576, Bodipy 581, Bodipy TR, Bodipy 630, VivoTag645, and Texas Red.
 8. The method of claim 1, wherein the image allowsfor selective detection of one or more tumor associated cells withinabout 1 cm from the surface of the tissue resection bed and excludesdeep tissue emission.
 9. The method of claim 1, wherein the subject is ahuman.
 10. The method of claim 1, wherein the single-cell resolutionimaging system comprises: (a) a light source; (b) an optical receptorconfigured to receive the light from the object; and (c) an imageprocessor.
 11. The method of claim 1, wherein the image processorcomprises a field of view (FOV) substantially greater than a diameter ofa cell of the object and an analysis resolution substantially matched tothe diameter of a cell of the object and configured to receive andanalyze the light corresponding to each cell in the FOV.
 12. The methodof claim 1, wherein the light source is an optical fiber bundle.
 13. Themethod of claim 1, wherein the imaging probe clears preferentially fromhealthy tissue leaving both tumor cells and tumor associatedinflammation cells labeled.